This application seeks support for continuation of a project which investigates the role of substrates of the T cell receptor stimulated protein tyrosine kinases. Work supported from this grant led to the identification and initial characterization of the SH2 domain containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76), as well as two SLP-76 associated molecules. Earlier work demonstrated that expression of SLP-76 is absolutely essential for T cell development beyond the double negative three (DNS) stage in the thymus. The overall goal of the current proposal is to establish the role of SLP-76 in T cell function beyond this developmental stage and to probe the mechanisms of action of this adapter protein in mature T cells. To address this goal we have generated a series of novel genetically altered mice which allow for temporal and lineage specific excision of wild type SLP-76 and expression of mutant variants of this protein. This proposal includes 3 specific aims. The first will address the binary question of whether or not SLP-76 is required for T cell function, starting at the double positive stage in the thymus, and then examining the role of SLP-76 in peripheral T cells. Experiments will specifically address the importance of this protein in various T cell subsets, in naTve, effector, memory, and regulatory T cell function and in the T cell response to pathogenic stimuli. The second specific aim will extend work from the first by addressing what regions of SLP-76 are most critical for its function in various T cell subsets and provide insight into the molecular interactions mediated by SLP-76 which are essential. Additionally, experiments will be performed to distinguish between the role of SLP-76 in immunoreceptor tyrosine based activation motif receptors versus integrins. The third specific aim will attempt to modulate SLP-76 localization in vivo in the hopes that immune cell responses may be altered for potential therapeutic benefits. It is anticipated that the work proposed for this project will provide new insights into how T lymphocytes, key elements of the immune system, respond to challenges within the body. It is hoped additionally that this work will provide new insights into how T cell responses may be modulated to help combat diseases characterized by defective or overexuberant immune cell function.